CN114247485A - Micro-fluidic chip for particle screening and separation - Google Patents
Micro-fluidic chip for particle screening and separation Download PDFInfo
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- CN114247485A CN114247485A CN202011027416.4A CN202011027416A CN114247485A CN 114247485 A CN114247485 A CN 114247485A CN 202011027416 A CN202011027416 A CN 202011027416A CN 114247485 A CN114247485 A CN 114247485A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
Abstract
The invention provides a micro-fluidic chip which can be used for screening single particles and forming liquid drop package for export. The microfluidic chip is connected with a liquid sample feeding device to form a microfluidic chip device for forming single particle-wrapped liquid drops, and the microfluidic chip device can further form a microfluidic operation system for forming single particle-wrapped liquid drops with a particle capturing device. The invention also provides a method for forming target single particle wrapped liquid drops in the microfluidic chip and respectively leading out the target single particle wrapped liquid drops.
Description
Technical Field
The invention relates to the technical field of microfluidics, in particular to a technology for forming single particle-coated droplets by using a microfluidic chip and leading out the droplets, which can be used in the fields of single cell screening, single cell separation, single cell sequencing, single cell morphological analysis, single cell culture, drug screening and the like.
Background
The microorganism is the most abundant species of the earth species, plays a very important role in the ecosystem, and is an indispensable ring in the synthesis, degradation and circulation of biomass. Meanwhile, microorganisms are also closely related to human health-the number of microorganisms in the human body is ten times that of the cells of the human body. However, to date, more than ninety percent of microorganisms have not been cultured under laboratory conditions. The phenotype identification, sorting and genotype analysis (namely ' single cell technology ') of single living cells can avoid the tedious incubation process of microorganisms and analyze the heterogeneity and the operation mechanism of the deepest ' level of a life system. The single cell technology for microorganisms has been faced with this technical challenge: how to nondestructively and precisely separate out single microbial cells.
The current technologies for realizing single cell separation of microorganisms mainly include fluorescence flow technology (FACS) and micromanipulator (eppendorf). However, there are technical limitations in that FACS requires fluorescent labeling of cells, and there are problems in that labeling is difficult or cell activity is affected after labeling, and FACS for separating microorganisms is expensive. The micro-manipulation technology is complex in single operation (precise control of the position of the capillary tip is required, and the needle insertion and the needle withdrawal are included), and the flux is low.
The research groups at home and abroad report a method for separating and obtaining microbial single cells by a microfluidic method or a laser ejection strategy. For example, F.Teng, et al, Nondestructive Identification and Accurate Isolation of Single Cells a Chip with Raman Optical Tweezers, anal.chem., used Optical Tweezers to draw Cells from a cell pool to a sorting pool, and then used a pipette to sort out Single Cells. However, the dragging distance of a single cell is several millimeters, long manipulation time is needed, and in addition, the adopted separation channel structure is wide, so that the whole channel cannot be imaged in a visual field, and the existence of non-target cells is easily ignored.
The article y.wang, et al, Raman Activated Cell Ejection for Isolation of Single Cells, anal.chem. provides a method of pulsed laser Ejection. However, before ejection, the cells need to be naturally dried on an ejection substrate, and the pulse laser generates a strong photo-thermal phenomenon on the drying substrate, which seriously affects the physiological activity of the separated cells.
Disclosure of Invention
In view of the above, the present invention provides a technique for separating and guiding out target single particles, and the present invention realizes the detection and capture of single target particles in a microfluidic chip and the transfer from the microfluidic chip to an external test tube.
The term "microparticle" as used herein refers to particles capable of being suspended in a non-organic phase (e.g., aqueous phase) and passing through the microfluidic chip of the present invention, and includes particles of biological origin and non-biological origin, such as eukaryotic cells, prokaryotic cells, unicellular organisms, viral particles, organelles, particles formed from biological macromolecules, drug particles, drug carrier particles, liposomes, polymer particles, and the like.
The invention provides a micro-fluidic chip, which comprises an oil storage pool, a sample storage pool, a micro-channel and a sample inlet, wherein two ends of the micro-channel are respectively communicated with the oil storage pool and the sample inlet, the sample storage pool is of a closed hollow three-dimensional structure, and an outlet of the sample storage pool is communicated with the micro-channel through a micro-channel branch.
The oil storage pool is of a hollow three-dimensional structure.
The surface of the oil storage pool is a hydrophobic oleophylic surface.
The top of the oil storage pool is of an opening structure.
In a preferred embodiment, the diameter of the oil storage pool is 4-14 mm, and the depth of the oil storage pool is 0.5-2 mm; preferably, the diameter of the oil storage pool is 6-10 mm, and the depth of the oil storage pool is 0.5-1.5 mm; more preferably, the diameter of the oil storage pool is 6-10 mm, and the depth is 1-1.5 mm.
The microchannel is of a cylinder structure.
In a preferred embodiment, the microchannel is a column structure.
In another preferred embodiment, the width of the micro-channel is 10-100 um, and the depth is 10-100 μm.
In another preferred embodiment, the width of the micro-channel is 10-30 μm and the depth is 10-30 μm.
In another preferred embodiment, the width of the micro-channel is 30-50 μm and the depth is 30-50 μm.
In another preferred embodiment, the width of the micro channel is 50 to 100 μm, and the depth is 50 to 100 μm.
In another preferred example, the upper and lower channel walls of the microchannel are transparent optical mirrors.
In another preferred embodiment, the depth of the microchannel branch is the same as the depth of the microchannel.
In a preferred embodiment, the sample storage pool is a sealed cylinder cavity.
In another preferred example, the diameter of the sample storage pool is 30-1000 μm; preferably, the diameter of the sample storage pool is 30-100 μm.
In another preferred embodiment, the depth of the sample reservoir is 300 to 500 μm.
In another preferred embodiment, the microfluidic chip further comprises at least one cavity, and the cavity is a closed hollow three-dimensional structure, is distributed around the sample storage pool, and is not communicated with the sample storage pool.
The volume of the cavity is 1-10 mu L (range).
In a preferred embodiment, the number of cavities is two or more.
In another preferred embodiment, the cavities are evenly distributed around the sample reservoir.
The material of the microfluidic chip is selected from but not limited to quartz, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), borosilicate glass and calcium fluoride.
In another aspect of the present invention, a microfluidic chip device for forming single particle-encapsulated droplets is provided, the device comprising the microfluidic chip provided in the first aspect of the present invention and a liquid sample injection device, the liquid sample injection device being in communication with the sample injection port.
The liquid sample injection device is selected from but not limited to: a gravity-driven adjusting sample feeding device, an injector, a peristaltic pump and an injection pump.
In a preferred embodiment, the gravity-driven adjustment sampling device comprises a height-adjustable sample holder, a sample container and a conduit, wherein the sample container is communicated with the sampling port through the conduit, and the sample container can move up and down on the height-adjustable sample holder.
In another preferred example, the sample container moves up and down in a manner of manual adjustment.
In another preferred example, the sample container moves up and down in a mode of electric adjustment.
In another preferred embodiment, the height-adjustable sample holder is designed as a slide rail, and the slide rail is provided with an electrically movable slide block for fixing the sample container; preferably, the height-adjustable sample rack further comprises a height adjustment controller, and the height adjustment controller controls the sliding block to move up and down on the sliding rail.
In another aspect of the present invention, a microfluidic operating system for forming a single particle-encapsulated droplet is provided, comprising a microfluidic chip or a microfluidic chip device provided by the present invention, and a particle capture device selected from optical tweezers.
The optical tweezers device adopted by the invention is the prior art in the field.
The trapping in the invention means that a particle trapping device including optical tweezers is used for fixing target particles, so that the target particles do not move along with the chip when the microfluidic chip is moved.
In another preferred example, the microfluidic operating system further comprises a sample detection device, including but not limited to a raman detection device, an optical microscope, a fluorescence microscope.
In another preferred embodiment, the microfluidic control system further comprises a device for guiding out the single particle-coated droplet, and the device for guiding out the single particle-coated droplet is selected from a capillary tube and a pipette tip.
In another aspect of the present invention, there is provided a method for forming and extracting a droplet encapsulated by a single particle, the method using the microfluidic chip or the microfluidic chip device or the microfluidic operating system provided by the present invention, and comprising the steps of: vacuumizing the microfluidic chip; injecting the particle phase solution into the outlet of the micro-channel communicated with the oil storage pool, and filling the sample storage pool with the particle phase solution by the negative pressure provided by the cavity; injecting the aqueous phase solution into the micro-channel through the sample inlet of the micro-fluidic chip to wash out the micro-particles until no particles remain in the whole micro-channel part and the particles in all the chips are positioned in the sample storage pool; taking the water phase flowing out of the oil storage pool, replacing the water phase with the oil phase, adjusting the height of the liquid sample injection device to balance the water phase in the micro-channel and the oil phase in the oil storage pool, and stopping the interface of the water phase and the oil phase at the position of the micro-channel close to the oil storage pool; capturing target particles by using a particle capturing device, dragging the target particles into the microchannel, raising the height of the liquid sample injection device to enable water in the microchannel to flow towards the oil storage pool, releasing the particle capturing device, enabling the target particles to enter the oil storage pool along with the water phase and forming liquid drops wrapped by single target particles; sixthly, guiding out the liquid drops wrapped with the single target particles.
The means for regulating the flow of liquid in the microchannel include, but are not limited to, gravity-driven regulation, syringe pump-driven regulation, peristaltic pump-driven regulation.
In a preferred embodiment, the particle capturing device captures target particles from the sample storage pool.
In another preferred example, the oil phase is selected from one or more of mineral oil, silicone oil, fluorocarbon oil, vegetable oil and petroleum ether.
In another preferred example, the particle phase contains particles and a liquid which is immiscible with the oil phase liquid in the oil reservoir; preferably, the particulate phase is a non-organic phase; more preferably, the particulate phase is an aqueous phase or a non-organic buffer.
In another preferred embodiment, the method for forming and discharging single particle-encapsulated droplets further comprises a sample detection step, wherein the sample detection step is performed before the fifth step, and the sample detection step is performed by a method selected from, but not limited to, raman spectroscopy, fluorescence detection, optical microscopy, and conductance detection;
in another preferred example, the method for guiding out the liquid drop wrapped with the single target particle comprises a capillary tube guiding method and a pipette guiding method;
in another preferred example, the method for forming and exporting single particle encapsulated droplets further comprises performing further operations on exported target particles, wherein the operations comprise single cell sequencing, single cell morphological analysis and single cell culture.
In another preferred embodiment, the method for collecting the characteristic signal of the single particle in the sample channel is selected from raman signal collection, fluorescence detection, optical microscope detection; preferably, the position for collecting the characteristic signal of the single particle is the detection pool.
In another preferred example, the laser wavelength of the optical tweezers is 1064 nm.
In another aspect of the invention, there is provided an application of a microfluidic chip, a microfluidic chip device or a microfluidic control system, including single particle screening, formation of a single particle-encapsulated droplet, or derivation of a single particle-encapsulated droplet.
In a preferred embodiment, the microparticles are cells, including but not limited to bacteria, fungi, mammalian cells.
The invention has the following technical advantages:
1. the method is suitable for enrichment detection of various sizes of particles, such as yeast cells with the size of tens of microns and bacterial cells with the size of about 1 micron.
2. Realizes the selection, separation and derivation of single cells, has low influence on the activity of the cells, and can be successfully docked with downstream single cell sequencing
3. The speed of screening and transferring single particles is high, and the operation time of transferring the single particles from the chip to the test tube is about 15 seconds.
4. The chip can be repeatedly used, and the operation cost is reduced.
5. The operation is simple.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic view of a microfluidic chip design;
FIG. 2 is a schematic diagram of sorting single cells on a microfluidic chip.
A. The cell suspension to be sorted is completely positioned in the sample storage pool (2).
B. The target cell is moved into the microchannel (3) by the optical tweezers.
C. The target cells enter the oil storage pool (1) along with the water phase to form single-cell liquid drops.
The main reference numbers: the device comprises an oil storage pool (1), a sample storage pool (2), a micro-channel (3), a sample inlet (4), a cavity (5) and a micro-channel branch (6).
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, but not all of the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1 preparation of microfluidic chip:
the design of the micro-fluidic chip is shown in the attached figure 1, and the channel design mainly comprises an oil storage pool (1), a sample storage pool (2), a micro-channel (3), a sample inlet (4), a cavity (5) and a micro-channel branch (6). Wherein the upper layer of the oil storage pool (1) is not provided with a cover plate and has a semi-open structure. The micro-channel (3) and the sample storage pool (2) are of closed structures. A plurality of closed cavities (5) are formed around the sample storage pool (2), and the cavities (5) are independent and are not communicated with the sample storage pool. The whole chip structure is prepared by a photoetching method in the traditional microfluidic field and is formed by bonding a layer of PDMS with a channel structure and a piece of double-layer mirror glass. The sample introduction device is a sample support with adjustable vertical height, and pure water is introduced into the chip microchannel through liquid level difference. The optical tweezers device adopts 1064nm laser.
The diameter of the oil storage pool (1) is 6-10 mm, and the depth is about 1 mm; the right side is communicated with a micro channel, the size of the micro channel (3) is about 10 to 100 mu m wide, and the depth is 10 to 100 mu m (the channel size is determined according to the sorted cells, the small-size cell of the bacterium uses a channel of 10 to 30 mu m, the medium-size cell of the yeast uses a channel of 30 to 50 mu m, and the mammalian cell generally uses a channel of 50 to 100 mu m); the micro-channel branch (6) is communicated with a sample storage pool (2), the volume of the sample storage pool is determined according to the quantity to be sorted, the sample storage pool is generally designed to be 100-1000 mu m, if the sample storage pool is used for single cell culture, the diameter of the sample storage pool is about 30-100 mu m, and sealed cavities (5) are distributed around the sample storage pool (2); the other end of the micro-channel (3) is communicated with a sample inlet (4).
The preparation method of the chip refers to the preparation method of the traditional PDMS microfluidic chip: and (5) making a chip structure diagram by using CAD software, and printing a mask. And preparing the SU-8 chip die according to a soft lithography method. PDMS was poured into a mold and cured at 80 ℃ for one hour. And taking the cured PDMS out of the mould, wherein the designed channel structure is left on the PDMS. And then bonding the PDMS layer and the glass through oxygen plasma bombardment to finish the preparation of the chip.
EXAMPLE 2 fluorescent Escherichia coli Single cell sorting
1. Before sorting begins, the fluorescent E.coli suspension is introduced into the sample reservoir (2) of the chip.
The air permeability of PDMS is utilized, the chip is firstly placed into a dryer with a vacuum cavity connected with a vacuum pump for vacuum pumping, the chip is taken out after a certain time, the cell suspension is spotted at the outlet of the micro-channel in the oil storage pool (1) by a pipette, and the cell suspension can slowly fill the sample storage pool (2) by means of the residual negative pressure of PDMS, particularly the negative pressure provided by the cavity (5), as shown in figure 2A. Then, pure water is connected into a sample inlet (4) of the chip to continuously wash the microchannel (3). Eventually, the entire microchannel (3) section will be free of residual cells, with all cells within the chip being located within the sample reservoir (2).
2. And taking the water phase in the oil storage pool (1) away and replacing the water phase with the oil phase. At the moment, the water phase in the micro channel (3) and the oil phase in the oil storage pool (1) are balanced by adjusting the height of the liquid sample introduction device, and the interface stays at the position of the micro channel (3) close to the oil storage pool (1).
3. And (5) starting sorting.
Fluorescent escherichia coli is green fluorescent protein, a fluorescent light source is turned on, and the shutter is switched to blue light excitation. Coli large cells emitting green fluorescence can be observed in the visual field. The target fluorescent escherichia coli single cells in the sample storage pool (2) are captured and dragged into the microchannel (3) by using an optical tweezers device, as shown in fig. 2B. The height of the sample injection device is raised, the optical tweezers are released, and the cells flow into the oil reservoir (1) along with the pure water phase and are wrapped by the generated water-in-oil droplets, as shown in fig. 2C. All the generated droplets were removed by capillary to complete single cell separation.
Through fluid mechanics calculation, the flow of the pure water phase in the micro-channel can not interfere with the sample storage pool communicated with the micro-channel, so that no other cells are introduced into the micro-channel filled with the pure water phase except for the target cells captured and dragged by the optical tweezers, and the success rate of separating single cells in the micro-channel is ensured.
Claims (10)
1. The utility model provides a micro-fluidic chip, its characterized in that, micro-fluidic chip includes oil storage pool, sample storage pool, microchannel and introduction port, the microchannel both ends communicate with oil storage pool and introduction port respectively, the sample storage pool is airtight cavity spatial structure, and the export of sample storage pool is passed through microchannel branch road and is communicated with the microchannel.
2. The microfluidic chip according to claim 1, wherein the oil reservoir has a hollow three-dimensional structure, an upper opening, and a hydrophobic and oleophilic surface.
3. The microfluidic chip according to claim 1, wherein the microchannel has a cylindrical structure, and the upper and lower channel walls are transparent optical mirrors.
4. The microfluidic chip according to claim 1, wherein the microfluidic chip further comprises at least one cavity, and the cavity is a closed hollow three-dimensional structure and is distributed around the sample reservoir and not communicated with the sample reservoir.
5. The microfluidic chip according to claim 4, wherein the number of the cavities is two or more and is uniformly distributed around the sample reservoir.
6. A microfluidic chip device, comprising the microfluidic chip of claim 1 and a liquid sample introduction device, wherein the liquid sample introduction device is in communication with the sample inlet.
7. A microfluidic operating system comprising the microfluidic chip of claim 1 or the microfluidic chip device of claim 6, and a particle capture device.
8. The microfluidic operating system of claim 7, wherein the particle trapping device is an optical tweezer.
9. The microfluidic operating system of claim 7, further comprising a sample detection device.
10. A method for forming and discharging single particle encapsulated droplets, wherein the method uses the microfluidic chip of claim 1 or the microfluidic chip device of claim 6 or the microfluidic operating system of claim 7, and comprises the steps of: vacuumizing the microfluidic chip; injecting the particle phase solution into the outlet of the micro-channel communicated with the oil storage pool, and filling the sample storage pool with the particle phase solution by the negative pressure provided by the cavity; injecting the aqueous phase solution into the micro-channel through a sample inlet of the micro-fluidic chip to wash the micro-channel until no particles remain in the whole micro-channel part and all the particles in the chip are positioned in the sample storage pool; taking the water phase flowing out of the oil storage pool, replacing the water phase with the oil phase, adjusting the height of the liquid sample injection device to balance the water phase in the micro-channel and the oil phase in the oil storage pool, and stopping the interface of the water phase and the oil phase at the position of the micro-channel close to the oil storage pool; capturing target particles by using a particle capturing device, dragging the target particles into the microchannel, raising the height of the liquid sample injection device to enable water in the microchannel to flow towards the oil storage pool, releasing the particle capturing device, enabling the target particles to enter the oil storage pool along with the water phase and forming liquid drops wrapped by single target particles; sixthly, guiding out the liquid drops wrapped with the single target particles.
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| CN202011027416.4A CN114247485B (en) | 2020-09-25 | 2020-09-25 | Micro-fluidic chip for particle screening and separation |
| PCT/CN2021/117046 WO2022062897A1 (en) | 2020-09-25 | 2021-09-07 | Microfluidic chip for particle screening and separation |
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